title: Native antigen fractionation protein microarrays for biomarker discovery.
abstract: In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.
authors.affiliation: Molecular Urology Laboratory, Division of Urology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Caiazzo
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: O'Rourke
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Barder
authors.forename: Bryce P
authors.initials: BP
authors.lastname: Nelson
authors.forename: Brian C-S
authors.initials: BC
authors.lastname: Liu
mesh_terms: D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D002457:Cell Extracts / Q000276:immunology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D019295:Computational Biology; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D000941:Antigens; D001323:Autoantibodies; D015415:Biomarkers; D002457:Cell Extracts; D005456:Fluorescent Dyes; D007074:Immunoglobulin G